Swine erysipelas vaccine



United States Patent 721 swmn nursuiunas VACCINE Dennis George Howell,Richmanswonth, Robert Parr, South 'Efliug, London, and KathleenRosemary'Heath,

Northolt, Greeu'r'ord, England, assignors to Glaxo" Group Limited, aBritish corporation No Drawing. Original No. 3,083,142, dated Mar. 26,1963, Ser. No. 795,322, Feb. 25, 1959. Application for reissue Mar. 16,1964, Ser. No. 362,544 Claims priority, application Great Britain, Feb.27, 1958, 6,396/58; Aug. 14, 1958, 26,186/58 9 Claims. (Cl. 167-78)Matter enclosed in heavy brackets [1 appears in the original patent butforms'no part of this reissue specification; matter printed in italicsindicates the additions made by reissue.

labour and hence expense involved in giving injections is considerableand to have to administer second or even third injections of vaccine mayrepresent a substantial increase in the cost ofrearing the animals. Thisproblem is particularly acute where range animals are concerned sincethe labour of rounding up the herds two or three times in the course ofa few weeks is often prohibitive.

It has been suggested that adjuvants could be added to vaccinepreparations for the purpose of delaying the release of antigen into thesystem and so thereby'reducing the number of doses necessary to set up asatisfactory antibody response. In particular, it has been proposed topresent the antigenic material in a water-in-oil emulsion, the antigenbeing contained in the disperse aqueous phase and its absorption intothe system so being delayed by the surrounding continuous oil phase.Such preparations have in many instances proved unsatisfactory in use asthey frequently give rise to considerable undesired local reactions atthe site of injection which may impede the desired release of antigen.

We have found that a vaccine dispersed in certain types of oil-in-watcremulsion hereinafter referred to possess marked advantages over thehitherto proposed water-in-oil emulsion-based vaccines. The oil-in-wateremulsions thus used for the purpose of the present invention are thosewhich can be described as being stabilised and remaining substantiallystable even following injection into muscle tissue. In experiments, wehave carried out, we find that many emulsions, when injected into livingmuscular tissue, rapidly break down into separate oil and Water phaseswith the result that the oil phase tends to cause necrosis or othertissue damage, whilst the separated aqueous phase no longer has theprotecting action of the oil intended to delay release of antigen; thisphenomenon is noted even with emulsions which in vitro would normally beconsidered as quite stable. For the purpose of the present invention,therefore, we use emulsions which are stablilised, i.e. so formulatedthat theyv remain substantially stable for some time following injectioninto anirnal' rnuscle tissue.

The vaccines prepared according to the invention cause substantiallyless tissue damage than vaccines hitherto prepared with oily adjuvantsand give rise to a more reliable and usually better delayed release ofantigen.

It has also been observed that vaccine preparations Re. 25,721 ReissuedJan. 26, 1965 according to the invention are in general easier to injectthan many hitherto proposcdvaccines based on water-inoil emulsions,being capable of production in a more readily flowing form.

According to the'present invention we provide an injectable vaccinepreparation comprising antigenic material dispersed in a parentcrallyacceptable stable emulsion of an oil dispersed in a continuous aqueousphase.

The term antigenic material is used in this specifica tion to meanmaterial derived from the culture of a pathogenic organism, whichmaterial on introduction into animal blood gives rise to antibodiesserving to protect against or combat infection.

The oil-in-water emulsions according to the invention will generally beprepared with the aid of parenterally acceptable wetting agents whichserve to form and stabilise the desired emulsion.

As is well known, the formation of an oil-in-water emulsion asdistinctfrorn a watcr-in-oil emulsion involves suitable choice ofwetting agent or agents, having regard to the relative proportions ofthe oil and Water phases and their exact nature. The consistency of anemulsion and hence its suitability for injection is also dependent onits constituents. Thus for the purpose of the present invention it isnecessary so to choose the constituents of the emulsion that anoil-in-watcr emulsion (as distinct from a water-in-oil emulsion) isformed and that its consistency is such that it can be injected. It isfurther necessary that the emulsion be stable and remain as an emulsionafter injection into muscle tissue.'

The wetting agents used in the preparation of the stabilised emulsionforming the base of the vaccines according to the invention arepreferably of the non-ionic type. With such wetting agents more reliablestability of the emulsion on injection is secured and there is lesstendency for the Wetting agent to be precipitated from the aqueous phaseby components of the antigen preparation than when a cationic or anionicwetting agent is used.

We have found it preferable to employ a two-component wetting agentsystem, one of the wetting agents serving primarily to form the emulsionand the second primarily to stabilise the emulsion once formed. As isknown, for example, the water-soluble or preferentially water solublewetting agents generally act as emulsifying agents whilst wetting orsurface active agents which are oil-soluble (or preferentially oilsoluble) are known generally to exerta stabilising effect on emulsionsand many different wetting agents are now offered on the market both foremulsifying and stabilising purposes.

The following types of wetting agents have been found to be particularlyuseful for the purpose of the present invention.

Type (a).P,olyhydric alcohols, or their anhydrides, esterified withfatty acids, for example, sorbitan monostearatc, propylene glycolmonostearate, diglycol monostearate, mannitan mono-oleate, sorbitansesqui'oleate, glyceryl' monostcarate, propylene glycol mono-olcatc,propylene glycol mono'laurate, diglycol monolaurate, diglycolmono-oleatc, diglycol monopalmitate, diglycol monoricinoleate,pentaerythritol monostearate, pentaerythritol distearate pentacrythritoldioleate, pentaerythritol glycerol oleate, pentaerythritoldioleostearate, low-molecular polyo-xyethylene oleate, glycerylmono-groundnut acid ester, po-lyglyceryl monostearate,polyglyceryloleate, glycol mcnosteara-te, glyceryl mono-olcate, glycerylmonopalrnitate and low-molecular polyoxyethylene monostearate.

The wetting agents of the above type (a) are, in general, oil-solubleand hence suitable as stabilising agents The following type, type (b),however, are generally water-soluble andso suitable as emulsf'yingagents.

Type (b).Ployoxyalkylene derivatives of esters of olyhydric alcohols,for example polyoxyethylene sorbitan lonostearate, polyoxyethylenepropylene glycol monoearate, polyoxyethylene glycol monolaurate,polyoxy- ;hylene glycol mono-oleate, polyoxyethylene glycollonostearate, polyoxyethylene glyceryl stearate, polyxyethylene sorbitanmonopalm-itate, polyoxyethylene soritan monolaurate, poly oxyethylenesorbitan trioleate 1d polyoxyethylene sorbitan mono-oleate.

Further types of Wetting agents which may be of use .clude thefollowing:

Type (c).\Vater soluble fatty acid esters of polyhylene glycol, forexample polyethylene glycol 300 diurate, polyethylene glycol 300monostearate, polyethyll6 glycol distearate and nonaethylene glycolmono-oleate, wer molecular polyglycol ricinoleate, lower molecular)lyglycol monocaprate-caprylate, lower molecular polyycol monolaurate.

Type (d).Fatty alcohols condensed with ethylene :ide e.g. the materialsold as Collone AC. or Abracol W. These wetting agents are water-solubleand hence itable as stabilising agents. The stabilising agents of type(a) are preferably used conjunction with the emulsifying agents of type(b). 1us, one especially suitable combination is sorbitan onostearatewith polyoxyethylene sorbitan monostearate 1ile another is propyleneglycol monostearate with poly- :yethylene monostearate, advantageouslycombined with glycol monostearate. The material Collone AC. is prefablyused in conjunction with sorbitan monostearate d/or low-molecularpolyoxyethylene monostearate. The exact proportion of Wetting agents tobe used ll depend on their nature. However, using a combinarn ofnon-ionic emulsifying and stabilising agents, we .d in general that from0.5 to 1.5% of emulsifying ent in combination with from 0.5 to 1.5% ofstabilising ent gives satisfactory results. The ratio of emulsifying cutto stabilising agent can thus vary between 1:3 and 1. It should be notedthat too great a proportion of :tting agent-either of the emulsifying orthe stabilising peis undesirable and may cause instability or eveneaking of the emulsion or phase reversal. The proportion of oil to wateris important and preferly the proportion of oil to water in the emulsionshould t be more than 65% but preferably not less than :sirably theproportion of oil to water should be within range of from 60%. Thecomposition of the llllSlOIl base should, however, be correlated withthe antity of water (if any) added with the antigen and final product,in order to be reliably stabilised, preftbly contains more than 10% ofoil. In order to determine the suitability of a given set of .ulsioncomponents for preparing a stable oil-in-water .ulsion of suitablethinness for injection one merely :nds the components in a homogeniserand leaves the .ulsion so formed to stand for a prolonged period. ,ytendency to separation or complete cracking can 11 be simply observed.Where there is doubt as to ether an oil-in-water or a water-in-oilemulsion has :n formed, one can add a dye which is soluble in only aphase and examine to see whether the disperse or continuous phase iscoloured. Alternatively one can ermine the conductivity of the emulsionwhich would farlower for a water-in-oil emulsion than for an -in-wateremulsion. The emulsion should be tested vivo; for example, it may beinjected into the muscle ue of an experimental animal e.g. a mouse, and,after he days, the animal sacrificed and the tissue surrounding site ofinjection examined. If the emulsion is suffi- 1tly stabilised thereshould be no substantial necrosis I usually there will be some of theemulsion still sent. .h'e oils which may be employed to prepare theoilvater emulsions may comprise vegetable oils, for exple arachis,olive, sesame, soya bean, castor and cotton- :1 oils as well as oilyesters e.g. ethyl oleate, dibutyl sebacate and isoamyl salicylate.However, we find that mineral oils of a pharmaceutical grade e.g. lightliquid paraflin, are to be preferred as they in general, andsurprisingly, cause even less tissue irritation than the vegetable oils.This we believe to be due to possible greater stability of mineral oilemulsions and hence less tendency for them to break after injection, thefine oil droplets being eventually dispersed by phagocytosis.

In the preparation of the vaccines according to the invention it isconvenient to prepare the oil-in-water emulsion by, for example,blending the oil, water and wetting agents in a homogeniser often atelevated temperature, for example at about C., and to mix this emulsionwith the antigenic material in the desired proportions.

The final vaccine must not contain unwanted organisms and it is thusnecessary to sterilise the various components of the vaccine. Where heatis used for sterilisation, the emulsion or other components must besterilised in the absence of the antigenic material, which would be destroyed by the high temperature. If the emulsion does not withstand heatsterilisation it is possible to sterilise the oily and aqueouscomponents before emulsification. In general, the oil may be sterilisedby heating to about C. for one hour while the aqueous medium may besterilised in the autoclave at 15 lbs. pressure for 30 minutes. Theemulsion should be cooled before mixing with the antigenic materialsince few antigens can with stand temperatures in excess of 40 C. andare best kept at room temperature or below. However, it is oftenpreferable to emulsify the oil with an aqueous suspension of theantigenic material, particularly where a high concentration of antigenicmaterial in the final vaccine is required. Thus if the antigen isobtainable only in the form of an aqueous suspension and the emulsion isprepared separately, it is necessary to add a substantial quantity ofaqueous suspension to the emulsion to obtain the required high antigenconcentration whichmay seriously alfect the stability of the emulsion.In such cases it is often possible to avoid the elevated temperaturesgenerally used in preparing the emulsions since, when a water solublewetting agent, such as polyoxyethylene monostearate, is added to theanitgen suspension and a more oil soluble wetting agent, such aspropylene glycol monostearate, to the oil, it is generally possible toprepare the emulsion by blending at room temperature.

It is also possible to prepare the oil-in-water emulsions from a gel ofthe oil. The gelling agent is usually a metal soap of a long chain fattyacid eg aluminium stearate, aluminium oleate, aluminium palmi-tate,lithium stearate, calcium oleate etc. In general it is possible to use asomewhat lower proportion of oil in the emulsion when a gel is used. Onesuitable gel comprises arachis oil containing between 1.5 and 2.5%aluminium stearate.

While the antigenic material incorporated in the preparations accordingto the invention may be of substantially any kind or in any form, thefollowing have been found particularly suitable for presentation withoil-inwater emulsions.

(1) Bacterial toxoids from which dead bacteria have been removed, forexample, toxoids, from Staphylococcus aureus. Clostridial toxoids, inparticular toxoids from C1. welchii, types B, C and D, which giveprotection in various combinations against lamb dysentery and pulpykidney disease and enterotoxaemia in sheep and toxoids from C1. tetaniwhich give protection against tetanus.

(2) Bacterial vaccines containing dead bacteria, for examplepreparations from Erysinelothrix rhusiopathiae which give protectionagainst erysipelas in pigs and turkeys, also Brucella, Salmonella andLeptospiral vaccines. (3) Viral vaccines, for example, Swine fevervirus, Foot and-Mouth virus, Loupin ill virus', Enzootic abortion virus,Infectious Larynys tracheitis virus, Newcastle disease virus, andInfectious Bronchitis virus.

It will be appreciated that it is often advantageous to administer to ananimal two vaccines giving protection against ditferent diseases andthat it is convenient to form.

combine. these into a single vaccine for simultaneous administration.Thus the toxoid from C1. welchii type D toxin, which protects againstpulp-y kidney diseasein lambs, can be combined with toxoid from Cl..tetani, which protects against tetanus, so as to protect young lambsagainst both these diseases.

The antigenic material may be incorporated into the oil-in-wateremulsion in substantially any convenient It may thus be a dried solid,if desired purified, an adsorbate on a parenterally acceptableadsorbant, for example aluminium phosphate, or aluminium hydroxide,pumice or kieselguhr or a suspension in a parenterally acceptableliquid; The final vaccine should, however, be pyrogen free and shouldcontain as little material as possible which gives rise to unwantedreaction in the body. The aqueous phase of the final vaccine is alsopreferably substantially isotonic, that is the sodium chlorideconconcentration in the aqueous phase is preferably about 0.85%, and itmay contain preservatives such as phenol or thiomersalate.

The invention will now be described with reference to two particularembodiments thereof. It will be understood that these embodiments whilstrepresenting vaccines which are of considerable use are only examples ofmany which can be prepared according to the invention.

VACCINE FOR THE CONTROL OF ERYSEPELAS IN- SWINE Erysipelas in swine iscaused by the organism Erysipelothrix rhusiopathae and hitherto vaccinesfor the immunisation of swine against the disease have been prepared byculturing the organism on suitable nutrient media, killing the organismand adsorbing the immunising antigens so obtained on a suitableadsorbent, e.g. alumina, which is then suitably formulated forinjection. The adsorbate of antigens has previously been proposed to beadministered in various media, the principal use in Great Britain havingbeen of simple aqueous suspensions; such preparations have usually beenof unsatisfactory potency, and have given only relatively shortimmunity. To overcome these difiiculties a vaccine has been proposedwhich is in the form of a water-in-oil emulsion but it was notacceptable due to the severe local reactions which occurred afterinjection and was, furthermore, difficult to inject.

We have found that a particularly effective vaccine preparation for thecontrol'of erysipelas in swine can be formulated comprising a stabilisedemulsion of a parenterally. acceptable oil in water, having dispersedtherein an adso-rbate of immunising antigenic material derived from theculture of Ery-sipelothrix rhusiopathae upon an injectable adsorbent.Such vaccines in accordance with the present invention can readily beprepared and have good antigenicity and are stable, readily injectable,do not, in general, give rise to-marked'reactions and give improvedprolongation of action as compared with simple aqueous suspensions of anadsorbate of the antigens. In particular they are surprisingly betterfrom various points of view, especially ease of injection, reducedtendency to cause reactions, etc., than erysipelas vaccines based onwater-in-oil emulsions previously proposed.

In the preparation of the vaccines according to the invention, it ispreferred to prepare separately an adsorbate of the antigenic materialand blend this with an emulsion of the chosen oil in water; preferablythe adsorbate of the antigens is first suspended in water containing abactericidal agent, e.g. formalin or thiomersalate, and made isotonicwith blood e.-g. withthe addition of sodium chloride. This suspensionisthen-blended with the emulsion;

Preferably the proportion of oil to water in the emulsion beforeaddition ofthe antigenic material is not more than 65% and isadvantageously within the range of from -60%.

The proportion. of adsorbate' isalso preferably withii the range offrom525%, conveniently 10%.

The adsorbate of the antigens. may be formed from an suitable adsorbent,examples of which are aluminum hy droxide, aluminum phosphate, pumiceand kieselguhr The adsorbate may be prepared in any desired way, fo:example by culturing the. organism on a suitable nutrien medium,inactivating the culture containing antigens witl for example formalinor thiomersalate, adding adsorben' to adsorb'the antigens, the resultantadsorbate being so arated, for example by centrifugation.

A virulent strain of organism should naturally be user for theproduction of this vaccine and, in general, organisms showing nosubstantial filamentous growth are suit able. We have found itpreferable to culture the organism at or about 30 C., instead of themore usuai 37 C., as in this way a more virulent culture is obtained.The medium used'for the culture organism may be of standard type.

VACCINE FOR THE CONTROL OF LEPTOSPIROSIE IN CATTLE AND OTHER ANIMALSLeptospirosis can be caused by the organism Leptospira pornona, thedisease occurring in two phases. In the first phase, which is the acutephase, the infected animal shows clinical manifestations of the diseasewith accompanying fever, haemoglobinuria, and loss of condition whichsometimes results in death. In the second phase the leptospira organismscolonize the kidney tubules and the animals, although apparently freefrom infection, become carriers, shedding viable leptospira organisms inthe urine. They are thus a source of infection to any susceptible animalin the vicinity.

We have found it possible to formulate a particularly useful vaccineactive against leptospirosis comprising a stabilised parenterallyacceptable oil-in-water emulsion containing antigenic material derivedfrom the culture 0 Leptospira pornona.

We find that by formulating the new vaccine in a stabilised oil-in-Wateremulsion a product is obtained which gives good protection against bothphases of'leptospirosis and has reasonably prolonged action. Thus ournew vaccine is more reliable and'effective than leptospirosis vaccinesformulated in various other vehicles. In particular the new vaccine ismore acceptable than a similar-product in the form of a water-in-oilemulsion.

The vaccine according to the invention is prepared by culturingLeptospira pomona, preferably a virulent strain thereof, in a culturemedia therefor, killing the organism and incorporating the antigenicmaterial thereby obtained into a s tabilised oil-in-water emulsionaccording to this invention. Preferably the Whole culture broth is usedin the formulation of the vaccine.

We have found furtherthat'thechoice of means used to kill the organismis'of importance'if best results are to be 0 tained. Thus, whilst forexample thiomersalate, formalin or heat may be used for this purpose,much-improvide results are obtained by the use of a phenol. Such phenolsinclude phenol itself, as well as the cresols resorcinol and thexyleno-ls, of which phenol is preferred.

Various media may be used to support the growth of Leptospira pomonaas-Willbe well known to those skilled in the art. As withallmicroorganisms the cultural requirements of'Leptospirapomonaessentially comprise a source of nitrogen, a source of carbonan'd'energy and nutrient salts. Very many materials are now known forthis purpose, so thatthe choice of nutrients is very Wide and it is asimple matter to choose nutrients suitable forv [he culture ispreferably carried out in at least two and ferably three stages. Forexample in the first or priry stage a culture is made from a suitablesource of organism e.g. an infected animal and grown up to m' a suitableinoculum for a secondary or development ge. The culture from thesecondary stage is then used inoculum for a production stage. The mediaused are iveniently of the same constitution for each stage.

a suitable temperature for the culture is 27 C. Following the culturethe organisms are killed and the )th is then suitable for formulation.

in the formation of the vaccine we prefer first to form oil-in-wateremulsion base and then incorporate the :igenic material therein carebeing taken to keep the .ulsion as an oil-in-water emulsion.

?referably the proportion of oil to water in the emuln and beforeaddition of the antigenic material is not re than 65% and isadvantageously within the range from 40-60%.

After formation of the emulsion base, the antigenic .t'erial preferablyas the whole culture broth is incorrted therein by any convenientmethod, preferably by z of a high speed blender or homogeniser. Theprortion of culture broth to be incorporated in the emul- 11 base ispreferably from 40 to 60% of the base.

if desired instead of first forming an emulsion base and :nincorporating the anhydrous material therein, the .ture broth may forexample be simply diluted as necary and the oil emulsified therein. Inthis case it is :ferable to add a water-soluble wetting agent to theueous suspension and an oil-soluble agent to the oil. The use of vaccineadjuvants, such as aluminium hy- )xide gel or aluminium phosphate is notnecessary acrding to the invention, but such adjuvants may if deed beused. Thus for example the antigenic material Ly be adsorbed on to somesuitable adsorbent and the sorbates incorporated into the emulsion base.The dit incorporation of the whole culture broth into the se is howeverpreferred.

For the better understanding of the invention we now 'e the followingexamples by way of illustration only.

Zxample 1.Preparation of Swine Erysipelas Vaccine (a) PREPARATION F THECULTURE BROTH ver extract (as above) 8%.

Z. amine (type E) 4% in distilled water. dium chloride 0.5%.

ucose 0.05%.

Heat to dissolve adjust the pH to 7.5. Filter while 11 hot throughWhatrnans No. 54 filter paper. Sterie by autoclaving at lbs. pressurefor minutes.

id sterile inactivated horse serum, rnls. per litre. cubate at 37 C. totest for sterility.

) THE PREPARATION OF ALUMINIUM HYDROXIDE This is prepared by the methoddescribed in the World :alth Organisation report No. 61. The gel isprecipited from aluminium sulphate by ammonia in the presce of ammoniumsulphate, washed free from soluble lts and sterilised by autoclaving.

The aluminium hydroxide content is calculated to eight in grams per ml.of gel.

((3) OIL-IN-WATER EMULSION This is a sterile liquid paraffin emulsionsyringeable rough a No. 26 (British standard wire gauge) needle eparedfrom:

the

Light liquid paraflin (medicinal grade) a 44.5% w./v. Grill 8(emulsifying agent) 1.1% w./v. Crill 3 (stabilising agent) 0.9% w./v.Sodium chloride 0.85% Vehicle Thiomersalate 0.015% to Distilled water to100% 100% Sterilise the parafiin by heating at 160 C. for one hour.Disperse the crills and dissolve the sodium chloride and thiomersalatein the water, then sterilise at 15 lbs. pressure for 30 minutes.

Add the water to the oil when both phases are at a temperature of 60 C.Homogenise. The final product contains of this emulsion.

(d) VACCINE PREPARATION Culture and incubation.-The strain employed forvaccine production is maintained in a freeze dried state (lyophilised)and when required is reconstituted in liver serum broth medium,incubated overnight at 30 C. and tested for purity. This culture is thenused to inoculate the amount of broth required as an inoculum for thevaccine.

An 18 hour liver serum broth culture is used, adding 10 mls. of inoculumto 1 litre of medium. This is incubated at 30 C. for 48 hours.

Killing of the bacteria.Add thiomersalate to the culture to give a finaldilution of 0.02% or formalin 0.25%. Leave the culture in the incubatorat 30 C. for 3 days and then test for any live bacteria by adding aquantity of the culture to a digest meat broth containing 0.1% cysteinehydrochloride to counteract the presence of any thiomersalate.

Adsorption.When the culture is proved to be completely dead, enoughaluminium hydroxide gel is added to give a final concentration of 1mgrn. per ml. The vaccine is shaken well and left overnight at +4 C. toadsorb.

Concentration.-The vaccine is now put through a centrifuge and thesupernatant fluid removed. The deposit is scraped from the bowl of thecentrifuge and thiomersalate saline is added. (The deposit from litresof culture is made up to 2 litres with thiomersalate saline. Thisconstituted 10% of the final vaccine.) A mechanical stirrer is used tobreak up the deposit, which is then added to the emulsion andhomogenised. The deposit, from 100 litres of culture being made up to 20litres with theemulsion so concentrating five times.

Example 2.Preparation of Leptospirosis Vaccine Culture medium: GramsBacto-peptone 0.8 Sodium chloride 1.4 Sodium bicarbonate 0.02 Potassiumchloride 0.04 Calcium chloride 0.04 Potassium dihydrogen phosphate 0.24Disodium hydrogen phosphate 0.69

Distilled Water to 1 litre.

The medium is boiled to dissolve the constituents, autoclaved to allowany phosphate to precipitate and filtered. The pH is adjusted to 7.2 andthe medium is bottled and autoclaved.

56 for two hours is added to the medium.

CULTIVATION OF VACCINE Before inoculation 10% sheep serum deactivated atThese flasks are incubated to maximum growth which is generally achievedin from seven to fourteen days.

Final vaccine cultures.T he leptospira are grown in 1 litre flaskscontaining 500 ml. of medium and 50' ml. of

serum. One secondary culture is added to each vaccine flask and thewhole incubated for seven days. Tests for sterility are carried out onthe fifth day. The cultures in all the flasks are pooled after sevendays growth. An aliquot is removed and counted. Phenol to 0.5% is thenadded to kill the leptospira and the pooled vaccine is refrigerated at4" C.

After seven days the vaccine is then mixed in 50:50 proportions with anoil-in-water emulsion prepared as follows:

OIL AND WATER EMULSION This is a sterile liquid paraffin emulsionsyringeable through a No. 26 (British standard wire gauge) needleprepared from:

Light liquid paraflin (medicinal grade) 44.5% w./v. Grill 8 (emulsifyingagent) 1.1% w./v. Grill 3 (stabilising agent) 0.9% w./v. Sodium chloride0.85 Thiomersalate 0.015% g Distilled water to 100% o 7 Example 3 PartsSorbit'an monostearate (stabilising agent) 1.5 Polyoxyethylene sorbitanmonostearate (emulsifying agent) 0.5 Light liquid paralfin 45.0 Saline53.0

Example 4 Polyoxyethylene monostearate (emulsifying agent) 0.5 Propyleneglycol monostearate (stabilising agent) 2.0 Diglycol monostearate 0.75Light liquid parafiin 43.75 Saline 53.0

In this example the light liquid parafiin may be replaced with an equalamount of arachis oil.

Example 5 Parts Sorbitan monostearate (stabilising agent) 2.0Polyoxyethylene sorbitan monostearate (emulsifying agent) 1.0 Aluminiumstearate/arachis oil gel (2.4%) 35.0 Saline n 62.0

We claim:

7 1. An injectible vaccine preparation in the form of an oil-in-wateremulsion comprising a preformed emulsion base which is stable in thepresence of body fluids upon intramuscular injection and is anoil-in-water emulsion containing an aqueous phase substantially isotonicto the blood and an oil phase consisting essentially of from 30% to 65%of mineral oil, from 0.5% to 1.5% of a watersoluble nonionic emulsifyingagent and from 0.5% to 1.5% of an oil-soluble nonionic stabilisingagent; and an antigenic material consisting of dead Erysipelothrix [rhusiopathol] rhusiopathae, the entire preparation containing more than 10%of mineral oil.

2. A preparation as claimed in claim 1 in which salt antigenic materialis an adsorbate on a parenterally ac ceptable adsorbent.

3. A preparation as claimed in claim 1 in which sair emulsifying agentis a polyoxyalkylene derivative of hexitan ester and said stabilisingagent is a fatty acid este: of a polyhydric alcohol.

4. A preparation as claimed in claim 1 in which sair emulsifying agentis polyoxyethylene sorbitan mono stearate and said stabilising agent issorbitan mono stearate.

5. A preparation as claimed in claim 1 in which thr proportion of oil towater in the emulsion base is betweei 40 to 60% p 6. A preparation asclaimed in claim 1 in which sair oil includes a gelling agent.

7. A preparation as claimed in claim 6 in which salt 1 gelling agent isa metal soap of a long chain fatty acid 8. An injectible vaccinepreparation in the form of a1 oil-in-water emulsion comprising apreformed emulsiot base which is stable in the presence of body fluidsMP0) intramuscular injection and is an oil-in-water emulsiol containingan aqueous phase substantially isotonic to tlu blood and an oil phaseconsisting essentially of from 30% to 65% of mineral oil, from 0.5% to1.5% of water-soluble nonionic emulsifying agent and from 0.5% to 2.0%of on oil-soluble nonionic stabilising agent; am an antigenic materialconsisting of dead Erysipelothri; rhusiopathae.

9. An injectible vaccine preparation in the form of at oil-in-wateremulsion comprising a preformed emulsiol base which isstuble in thepresence of body fluids upo-t intramuscular injection and is anoil-in-water emulsiol containing an aqueous phase substantially isotonicto tht blood and an oil phase consisting essentially of iron 30% to 65%of mineral oil, from 0.5% to 1.5% of t water-soluble nonionicemulsifying agent and from 0.9% to 2.0% of an oil-soluble nonionicstabilising agent; am an antigenic material consisting of deadErysipelothri: rhusiopathae.

References Cited by the Examiner The following references, cited by theExaminer, arc of record in the patented file of this patent or theorigina patent.

UNITED STATES PATENTS 2,057,623 10/36 Beard 167-7 2,529,461 11/50Schneiderwirth 1677E 2,675,343 4/54 Clymer 16782 2,756,176 7/56 Maureret a1. 1677E FOREIGN PATENTS 425,406 3/35 Great Britain.

OTHER REFERENCES Freund: Annual Review of Microbiology, vol. 1 1947 (pp.291-308, page 304 is particularly pertinent) Annual Reviews, Inc.,Stanford, Calif.

Hoag et al.: Am. Jour. Vet. Res., 16 (60), pp. 381-385 (1955).

Trim et al.: Surface Activity and Permeability at Factors in DrugAction, pp. 111-142, in Symposia of the Society For ExperimentalBiology, No. III, Selective Toxicity and Antibiotics, 1949, AcademicPress, Inc. New York, N.Y.

Woodhour et al.: Development and Application 01 New Parenteral Adjuvantsv. Comparative Potencies ol Influenza Vaccines Emulsified in VariousOils, 1. Immunology, vol. 86, No. 6, pp. 681-689 (1961).

LEWIS GOTTS, Primary Examiner.

